The goal of this project is to isolate and characterize several vital enzymes involved in the post-transcriptional processing and modification of eukaryotic viral and cellular messenger RNA. Information gained from these studies related to the reaction mechanism, substrate specificity, and biological function, will significantly enhance the current state of knowledge and understanding of eukaryote gene expression and regulation. The study will be developed at two levels: (1) an original basic research investigation of several RNA processing enzymes responsible for the formation of the capped 5'-end of mRNA and the internal adenine methylation of messenger RNA; (2) an advanced isolation and comparative characterization studies on two modification enzymes, responsible for the additional methylation of the cap and the addition of methyl residues to the 5-position of cytosine. Enzymes will be isolated from various tissues utilizing state-of-the art techniques including ion exchange and affinity liquid chromatography. Purification and enzyme assay procedures as well as a variety of substrates will be developed for both the cap specific enzymes and those involved in internal methylation. The development of these substrates involves chemical, enzymatic and recombinant DNA procedures. Preliminary experiments have demonstrated the capping and methyltransferase activities in wheat germ and HeLa cells. Partial purification schemes and reasonable assay procedures have also been developed. The specific goals of this research project will be to define the biological parameters of these enzymes, including: isolation, physical characterization, substrate specificity, and characteristics of the catalyzed reaction and its intended biological function. From these data it may be possible to deduce a portion of the molecular logic involved in the regulation of gene expression.